Intraocular transplantation of encapsulated cells

ABSTRACT

Compositions and methods for reducing ocular diseases by implanting in an eye of a subject a composition comprising encapsulated cells which produce polypeptides, more particularly polypeptides that exhibit neurotrophic and/or anti-angiogenic activity. The encapsulation prevents the entry of host immune cells in the microcapsule while permitting the release of the polypeptide outside of the microcapsule.

This application is a divisional of application Ser. No. 09/449,293 nowU.S. Pat. No. 6,267,554, filed Nov. 24, 1999, which application(s) areincorporated herein by reference.

FIELD OF THE INVENTION

The present invention relates to compositions and methods for thedelivery of biologically active molecules to the eye. The inventionrelates more particularly to encapsulated cells, their preparation anduse to deliver in vivo effective amounts of biologically activemolecules. The invention is particularly suited for the treatment ofphotoreceptor degeneration retinopathies or cancers.

BACKGROUND AND PRIOR ART

There are many diseases of the eyes or ocular diseases which affectvision. Some of these diseases include diseases of the conjunctiva andcornea which can produce loss of visual acuity, pain and discharge,cataracts resulting in blurred vision, glare altered color perceptionand monocular diplopia, uveal diseases resulting in photophobia, oculardiscomfort and visual blurring, retinal diseases resulting in distortionof straight lines (metamorphopsia), loss of central acuity and visualfield abnormalities and diseases of Bruch's membrane, which may lead toage-related macular degeneration.

Among numerous forms of human retinopathies, age-related maculardegeneration (hereinafter referred to as AMD) is the leading cause ofsevere vision loss in the elderly and affects between 6.4% (New Zealand)(19) and 6.9-11.4% (U.S.A.) (20, 21) of people beyond the age of 65. AMDis a heterogeneous group of retinal degenerations which is associatedwith alterations of retinal pigment epithelium (RPE) cells.

In the past transplantation of rod-enriched adult photoreceptor cellpopulations that may protect the macula from secondary degeneration inrod-cone retinitis pigmentosa affected patients was generally thetherapy of choice for retinal degenerations, including AMD. However, thephotoreceptors that were to be transplanted usually came from adult orembryonic human photoreceptors collected post-mortem. Thesephotoreceptors can thus be infected by a diversity of viruses includingthe hepatitis virus, cytomegalovirus, and human immunodeficiency virusas well as other pathogens such as prions that may remain undetectableprior to transplantation. Additionally, these cells may trigger a strongimmune response in the host, leading to the destruction of the graftand/or to inflammatory autoimmune reactions.

Moreover, oculo gene therapy protocols which relied on the basis of celltransfection, viral cell infection, or cell transplantation were alsoknown in this art. However one problem with the known gene therapyprotocols was that adverse immunological side effects occurred. Theseadverse side effects, in some instances, could lead to anaphylacticshock, endangering the patient.

Among various protocols which have been designed to transfertherapeutically important genes to a target tissue, the transplantationof genetically engineered cells has become particularly promising inrecent years. To protect allogeneic or xenogeneic grafts from immunerejection, cells have been successfully microencapsulated withnon-degradable biocompatible polymers and used for the experimentaltreatment of several disorders such as type I diabetes mellitus (1-5),dwarfism (6), hemophilia B (7), anemia (8), chronic pain (9, 10),amyotrophic lateral sclerosis (11), as well as Parkinson's,Huntington's, and Alzheimer's diseases (12-18). However, none of thesecited references taught a method for treating ocular diseases.

Thus, it is an object of the present invention to provide geneticallymodified cells which stably produce therapeutic factors suitable fortreating ocular disorders.

It is another object of the present invention to provide methods ofencapsulating such genetically modified cells, in such a way that thecells remain viable and produce effective amounts of the biologicallyactive agent(s) in the eye, more particularly in the retina.

It is another object of the present invention to provide a method forimplanting in vivo these devices (encapsulated cells), and the effectiverelease and treatment of ocular disorders in vivo.

It is another object of the present invention to provide ocularcompositions that can be surgically retrieved from the ocular globes,for instance, in case of an occurrence of adverse side effects.

It is yet another object of the present invention to providecompositions and methods to deliver biologically active molecules to theeye to treat ocular disorders that are bacteriological and virologicalsafe.

It is yet another object of the present invention to provide geneticallyengineered cell lines that can be systematically tested for any kind ofpathogens before they are encapsulated and transplanted into human eyes.

It is yet another object of the present invention to provide atherapeutic method to promote the survival of photoreceptor cells indegenerating retinas of mammals in order to treat retinal degenerations,as well as treating mammals that have retinitis pigmentosa orage-related macular degeneration.

These and other objects are achieved by the present invention asevidenced by the summary of the invention, description of the preferredembodiments and the claims.

SUMMARY OF THE INVENTION

In one of the method aspects, the present invention provides a methodfor delivering a polypeptide to an ocular cell of a subject in vivo saidmethod comprising the step of:

(a) implanting in an eye of said subject a composition comprising cellswhich produce said polypeptide wherein said cells are encapsulated.

In another method aspect, the present invention provides a method forreducing an ocular disease comprising the step of:

(a) administering to a subject in need of said treatment a compositioncomprising encapsulated cells wherein said cells produce a biologicallyactive polypeptide for reducing said ocular disease.

In a composition aspect, the present invention provides a compositioncomprising encapsulated cells which produce an endostatin or afibroblast growth factor polypeptide.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 are photographs of the microencapsulation and transplantation ofencapsulated NIH3T3 PS-FGF18 cells to the eye of a RCS p+ rat. A-B)Cells are packed into a 1.5 mm microcapsule and kept in culture for 1day (1) and 14 days (2) prior to transplantation. As seen through thelens, a 1.5 mm microcapsule (arrow) was implanted into the vitreouscavity (3). (4) is the explanted 1.5 mm microcapsule after being in thevitreous cavity for 90 days. Microcapsule diameter: 300 μm. Scale bars:200 μm (1, 2), 1 mm (3), 100 μm (4).

FIG. 2. is a graphic representation of the survival rate of NIH3T3-P16and NIH3T3 PS-FGF18 cells in 4.0 mm microcapsules, as measured at 2, 15,30, and 60 days post-encapsulation. Viable cells were identified bytrypan blue exclusion assays and are indicated as the mean percentage ofthe total number of encapsulated cells. Bars: s.e.m. (n=6).

FIG. 3. is a Western blot of crude cell extracts (105 cells/lane)revealing the 18 kDa hFGF-2 protein in NIH3T3 PS-FGF18 cells (lane I,arrow head). Lane II is an NIH3T3-P16 cell extract. Lane III isrecombinant hFGF-2 (10 ng).

FIG. 4 is a graphical representation of the secretion of hFGF-2 proteinby NIH3T3 PS-FGF18 cells at different concentrations. Data are expressedas mean values (pg/24 h)±s.e.m. (n=6).

FIG. 5 is a graphical representation of a bioassay of hFGF-2 secretionfrom 1.6 mm and 4.0 mm capsules filled with either NIH3T3-P16 or NIH3T3PS-FGF18 cells and kept in culture for 15, 30, or 60 days. Data areexpressed as mean values±s.e.m. Statistics: Mann-Whitney U-test(*P=0.001, n=6).

FIG. 6 are photographs of stained retinal tissues showing the retina ofa 21-day-old rat before the beginning of photoreceptor cell degeneration(1). A dystrophic retina of a 63 day-old control rat eye (2).Photoreceptor cell rescue in a rat eye which received microencapsulatedNIH3T3 PS-FGF18 cells and cut at 90 days post-transplantation (3). Notethe microcapsule-proximal retina which comprises 5 to 6 photoreceptorcell layers (arrow). The retina of an ocular globe proximal to aNIH3T3P16 cell-filled microcapsule, cut at 90 days post-transplantation(4). The photoreceptor cell layer is indicated by arrows. Sections: 16mm, toluidine blue staining. Scale bar: 20 mm.

FIG. 7 is a two-dimensional reconstruction of the retinal surface oftreated RCS p+ rats showing maximal rescue effects. The retinal areawith at least 5 photoreceptor cell layers is indicated in black. Theregion (4.10 mm²) of delayed photoreceptor cell degeneration in theretina exposed for 45 days to a NIH3T3 PS-FGF18 cell-filledmicrocapsules indicated in (1). The area (0.15 mm²) of delayed celldegeneration proximal to a NIH3T3-P16 cell-filled microcapsule as seenin the only eye which revealed a cell rescue effect at 90 dayspost-transplantation is indicated in (2).

FIG. 8 is a photograph of a photoreceptor rosette formation in a RCS ratretina at 45 days post-transplantation. The microcapsule containingNIH3T3-P16 fibroblasts (asterisk) caused a cellular reorganization ofthe retina which led to the appearance of a photoreceptor rosettestructure (arrow). The section (16 mm) was stained with toluidine blue.Scale bar: 40 mm.

FIG. 9 are photographs of tissue sections of rat retinas. Photoreceptorsare indicated by the arrows and retinal-pigment epithelial cells arerepresented by the asterisks. Normal 3-month-old RCS rdy+ albino areindicated in (1) and (2) and dystrophic 111-day-old RCS rat retina areindicated in (3) and (4). DAB-immunostaining without primary antibody(1; control) and with anti-FGF-2 antibodies (2-4). Note the lack ofimmunostaining in photoreceptor cells in the rescued retina (4).Cytoplasmic FGF-2 immunostaining in encapsulated NIH3T3 PS-FGF18fibroblasts at 90 days post-transplantation is represented in (5).Antibody concentration: 10 ng/ml; parafin sections countercolorated withhematoxyline: 5 mm. Scale bar: 10 mm.

FIG. 10 is a graphical representation of plasmid pRCEN1-Endo.

FIG. 11 are nucleotide sequences of the peptide signal VEGF (SEQ IDNO:1), hemaglutinine (SEQ ID NO:2) and endostatin used to construct theplasmid pRCEN1-Endo (SEQ ID NO:3).

FIG. 12 is a Western blot of CHO cells transfected with pRCEN1-endoafter TCA precipitation.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS OF THE PRESENTINVENTION

As used herein the term “subject” refers to man or any animal that haseyes.

As used herein “biologically active”, when referring to the polypeptidemeans that the polypeptide is capable of functioning.

As used herein “ocular disease” refers to a disease of the eye,including, but not limited to tumors, ocular degeneration,retinopathies, retinitis, retinal vasculopathies, diabeticretinopathies, diseases of the Bruch's membrane and the like.

As used herein, the term “reducing” also encompasses treating andalleviating the ocular disease.

More specifically, the present invention entails a method of deliveringa polypeptide to an ocular cell in vivo comprising implanting, in theeye of a subject, a composition comprising cells which produce saidpolypeptide wherein said cells are encapsulated.

The invention also entails the use of encapsulated cells for thedelivery of polypeptides in the ocular and more particularly the retina.

The instant invention can be used to deliver various polypeptides in theeye. In the context of this invention, the term “polypeptide” designatesany protein or fragment thereof, peptide or any molecule composed of anamino acid sequence, preferably having biological activity. Preferredbiologically active polypeptides according to the instant inventionexhibit neurotrophic and/or anti-angiogenic activity. Typical examplesof such polypeptides are ciliary neurotrophic factor (CNTF),glial-derived neurotrophic factor (GDNF), nerve growth factor (NGF),brain-derived neurotrophic factor (BDNF), NT3, nurturin, fibroblastgrowth factors (FGFs), endostatin, ATF, fragments of thrombospondin,variants thereof and the like. More preferred polypeptides are FGFs,such as acidic FGF (aFGF), basic FGF (bFGF), FGF-1 and FGF-2 andendostatin.

For use in mammalian subjects, it is preferred to use allogenicpolypeptides. In particular, for use in human subjects, it is preferredto use encapsulated cells which produce a polypeptide of human origin.Obviously, it is also possible to deliver xenogenic polypeptides orvariants thereof. In a specific example, the polypeptide is human FGF-2or human endostatin or variants thereof. The term “variant” means anypolypeptide having one or several structural modification(s) introducedtherein, such as mutation(s), deletion(s), substitution(s) and/oraddition(s) of one or several amino acid residues, the variant stillexhibiting biological activity. The use of the instant invention todeliver any such variants can be performed by the skilled artisanfollowing the teaching of the instant invention.

The compositions and methods of this invention use cells which producethe polypeptide. The cells can be primary cells or cell lines, inparticular genetically modified primary cells or cell lines whichproduce the polypeptide. Preferably, the cells are eucaryotic cells,more preferably mammalian cells. The use of mammalian cells isadvantageous since mammalian cells can produce polypeptides withappropriate post-translational modifications (glycosylations,maturation, etc.). The cells are preferably compatible with biologicaluse in vivo and, in particular, do not exhibit known pathogenicactivity. Examples of such cells include, but are not limited to,fibroblasts, muscle cells, hepatocytes, neural cells and the like, CHOcells, kidney cells, PC12 cell lines, MDCK cells, astrocytes, and thelike.

It is particularly preferred to use cells which can proliferate, atleast under certain culture conditions, in order to produce largeramounts of the polypeptide(s). Furthermore, the cells can be autologous(from the same subject), allogeneic (from a different subject of thesame species) or xenogenic (from a different species). Indeed, since thecells are encapsulated prior to their administration, they are protectedfrom the immune system of the receiving subject and can survive for longperiods of time.

Specific examples of cells to be used in the instant invention includefibroblasts, such as murine fibroblasts, in particular NIH-3T3 cells; C2cells, C12 cells, CHO cells, PC12 cells, and the like. In a preferredembodiment of this invention, fibroblast cells are used, in particularfibroblast cell lines.

At least some of the cells used in this invention produce a polypeptideto be delivered in the eye. Generally, the cells are geneticallymodified cells, i.e., they contain a recombinant nucleic acid moleculeencoding the polypeptide. The nucleic acid construct encoding thepolypeptide can be a DNA construct, in particular a cDNA or syntheticDNA and can be further modified to improve transcription/translation ina host cell. The nucleic acid can also be a RNA, for instance, aretroviral genome.

Preferably, the nucleic acid construct comprises, operably linked, apromoter region, a sequence encoding the polypeptide, and optionally, atermination signal. Even more preferably, the nucleic acid constructcomprises a secretion signal, between the promoter and coding regions,which allows, or facilitates, the secretion of the polypeptide outsideof the cells. The secretion signal may be homologous with respect to thepolypeptide (i.e., from the same gene) or heterologous thereto (i.e.,from any other gene encoding a secreted polypeptide, in particular amammalian gene, or artificial). Examples of secretion signals includethe signal peptide of vascular endothelial growth factor (VEGF), pre proNerve Growth Sequence (NGS) and the like.

The promoter region can be chosen from among all promoter regions thatare functional in mammalian cells, in particular human cells. Thepromoter can be a strong or a weak promoter, a constitutive or aregulated/inducible promoter, an ubiquitous or selective promoter. Thepromoter can be of different origin such as cellular, viral, artificialand the like. Particular types of promoters are house-keeping promoters,i.e., promoters from cellular genes expressed in mammalian (e.g., human)tissues or cells, or viral promoters (CMV, LTR, SV40, etc.) Furthermore,the promoter region can be modified artificially to include enhancerelement(s), inducibility element(s) and the like. The promoter,secretion signal and termination region sequences can be selected andadapted by the skilled artisan based on the polypeptide, the pathology,the vector used, etc. In this regard, the nucleic acid construct can beinserted in any various kinds of vectors such as plasmids, viruses,episomes, phages, artificial chromosomes and the like.

The cell composition can be prepared by contacting a cell culture with anucleic acid construct, as defined above, encoding the polypeptide. Thecells are then recovered and a selection step can be performed in orderto isolate the cells which effectively contain the recombinant nucleicacid construct and express the polypeptide. The contacting can be madeaccording to various methods known in the art and includeelectroporation, precipitation, gene gun, viral transduction, non-viralvector mediated transfection and the like. Preferably, the cells arecontacted with the nucleic acid construct without a viral vector. Inthis regard, the nucleic acid construct can be inserted in a non-viralvector (a plasmid, cosmid, and the like) and contacted with the cellpopulation either alone (“naked”) or in the presence of a transfectingagent (liposome, polymer, cationic lipid and the like). The cells arethen cultured according to known methods in order to provide sufficientamounts of transfected cells (usually above 10⁴ cells, preferably above10⁵ cells). The cells can be used directly or stored for later use.

In a preferred embodiment, stable cell lines producing the polypeptideare prepared. These cells usually contain one or several copies of thenucleic acid construct incorporated into their genome, so that theprogeny, daughter and/or derivatives of said cells still contain thenucleic acid construct and produce the polypeptide. The preparation ofsuch cells is advantageous since they can be used in many differentsubjects where the polypeptide is to be produced. These cell lines cantherefore be produced, cell banks can be made under appropriate qualityconditions and stored.

In a preferred embodiment of this invention, a composition is usedcomprising mammalian cells containing a recombinant nucleic acidconstruct, said cells producing the polypeptide. Particularly preferredembodiments of this invention use a composition comprising mammaliancells containing (i) a recombinant nucleic acid construct encoding anendostatin polypeptide, said cells producing the endostatin polypeptideor (ii) a recombinant nucleic acid construct encoding a fibroblastgrowth factor, said cells producing the fibroblast growth factor. In amore preferred embodiment, the composition comprises geneticallymodified fibroblast cells producing the polypeptide.

As indicated, above, the methods and compositions of this invention useencapsulated cells. The term “encapsulated” means that the cells arecontained in a biocompatible device or jacket which immunoisolates thecells. The cells are therefore preferably encapsulated in abiocompatible jacket which protects the encapsulated cells from theimmune cells and allows the release of the polypeptide outside of themicrocapsule. The main characteristics of the biocompatible materialused for encapsulation should therefore be as follows:

(i) it does not significantly activate the complement system;

(ii) it allows the release of the polypeptide outside of themicrocapsule; and

(iii) it significantly prevents the entry of the host immune cells inthe microcapsule.

Preferably, the biocompatible material used is a permeable orsemi-permeable material with pores having an internal diameter below 400μm, more preferably below 300 μm. Various biocompatible artificialpolymers (or copolymers) can be employed for encapsulation, derived fromcellulose, dextran, polyamide, polyurethane, acrylonitril, nylon,alginate-poly-L-lysine, hydroxyethyl methacrylate (HEMA), hydroxyethylmethacrylate-methyl methacrylate (HEMA-MMA), agarose-polystyrenealginate, etc. (2, 5, 6, 10-12, 14, 15, 17, 18, 34-42). In a specificembodiment, the copolymer AN69 (polyacrylonitril-methallylsulfonate) isused. The results presented in the examples show that this biopolymerprovides efficient protection of encapsulated cells against the hostimmune system and long-term maintenance of cell viability. AN69 can beused as a semipermeable hemodialysis membrane and fails to activate thecomplement system. Other biocompatible materials include hydrogels andthermoplastic materials, as described in, for instance, U.S. Pat. No.5,874,099, incorporated herein by reference.

The microcapsule or “jacket” can exhibit different forms. In particularembodiments of this invention, the microcapsule is shaped to form afiber (in particular, a hollow fiber) or a sheet (in particular a flator curved sheet). The size of the microcapsule which is usually below1.0 cm long or its diameter can be adapted by the skilled artisan.

Preferably, the composition comprises at least 10⁴ encapsulated cells,more preferably at least 10⁵ cells. The number of encapsulated cells canbe adapted by the skilled artisan depending on the cell type, thepolypeptide, the size of the microcapsule, etc. For encapsidation thecells are deposited on the biomaterial in any appropriate medium. Thecells, mounted by capillarity thus adhere to the biopolymer and canproliferate inside the microcapsules under culture and in oculoconditions, thus growing to confluence.

The composition can be implanted in different sites of the eye,preferably in the vitreous body of the eye, for instance in theintravitreus cavity, ocular globe or any other area which allows theinjection of a device according to the invention and contains injuredtissue. A preferred implantation route is the injection in theintravitreous cavity, especially for treatment of degenerative eyedisorders.

In this regard, the invention also provides a method of reducing anocular disease comprising administering intraocularly to a subject inneed of such treatment a composition comprising encapsulated cells whichproduce a polypeptide biologically active for reducing the oculardisease. The polypeptide is preferably a neurotrophic or anti-angiogenicpolypeptide as defined above. The ocular disease can be any oculardisease such as a tumor or tumors, ocular degeneration, retinopathies,retinitis and the like.

In a preferred embodiment, age-related macular degeneration (AMD) can bereduced using the methods and composition of the present invention. Thishas been demonstrated herein using the congenic Royal College ofSurgeons (RCS) rat strain which represents the closest and mostrecognized rodent model for retinal pigment epithelium (RPE) linkedretinopathies (22, 23). RCS rats have a recessive hereditary retinaldystrophy evident by the third postnatal week. This dystrophy ischaracterized by a progressive degeneration of RPE cells and a secondaryloss of photoreceptor cells by apoptosis (24, 25). In the dark-eyed ratstrain RCS p+, retinal dystrophy progresses to a complete photoreceptorcell degeneration at the age of 2 months (22, 23, 26). Cell degenerationis related to a deficiency of the phagocytosis of photoreceptor outersegments by RPE cells and is accompanied by a significant decrease inthe production of the basic fibroblast growth factor (FGF-2; 18 kDa) inthe retina (27). The results presented in the examples demonstrate thatthe instant invention can be used to treat, reduce or alleviatephotoreceptor degeneration and AMD. In particular, a significant rescueof photoreceptor cells is achieved in vivo upon intraocularadministration of the composition of the present invention. The capacityof the encapsulated cells of this invention to rescue photoreceptorcells was further demonstrated in the dystrophic RCS retina.

In another particular embodiment, the present invention thus provides anefficient method of reducing photoreceptor cells degeneration comprisingadministering intraocularly to a subject in need of such treatment acomposition comprising encapsulated cells which produce a biologicallyactive anti-angiogenic polypeptide. Preferably, the biologically activeanti-angiogenic polypeptide is an endostatin polypeptide or an FGFpolypeptide.

Other retinopathies include, for instance, circinate retinopathy,diabetic retinopathy, hypertensive retinopathy, leukemic retinopathy,pigmentary retinopathy, sickle cell retinopathy and the like, which canalso be reduced using the present invention.

The invention also relates to a composition comprising encapsulatedcells which produce an endostatin or an FGF polypeptide. Morepreferably, some of the encapsulated cells contain a recombinant nucleicacid construct encoding an endostatin or an FGF polypeptide, preferablya human endostatin or a human FGF. As explained above, the compositioncomprises preferably at least 10⁴ cells encapsulated in a biocompatiblejacket. A particular example is represented by a composition comprisingcells containing a nucleic acid construct encoding human FGF-2. In thisrespect, the cytoplasmic 18 kDa form of FGF-2 can be secreted despitethe absence of a hydrophobic secretory signal sequence. This protein isexported presumably via a mechanism of exocytosis independently of theendoplasmic reticulum-Golgi complex pathway (28-31). FGF-2 is eitherstored extracellularly by associating with heparin sulfate proteoglycansor binds with high affinity to membranous tyrosine kinase receptorswhere it exerts pleiotropic effects on cell growth, differentiation,morphogenesis, and cell repair (32). As demonstrated previously, humanFGF-2 (hFGF-2) can transiently delay photoreceptor cell degenerationwhen injected intravitreally or subretinally (33).

The invention thus provides compositions or methods which allow theintraocular production and secretion of this trophic factor byencapsulated cells, in particular mouse fibroblasts.

The invention shows, for the first time, that transplantation ofxenogeneic polypeptide-secreting cells, encapsulated in a biopolymer,into the vitreous body can promote the survival of photoreceptor cellsin the retina in vivo. As compared to other cell therapy protocols, thistechnique has the advantage to avoid possible implant-linked adverseside effects on the retina by surgically retrieving obsoletemicrocapsules. The transfer of encapsulated cells producing trophicfactors thus represents a new approach to treat, reduce or alleviateneurodegenerative retinopathies. The invention can also be used todeliver biologically active polypeptides to animals, in order to studytheir properties (stability, activity, etc.) or metabolism, or as atreatment.

Other advantages and embodiments of this invention will be disclosed inmore details in the following experimental section, which should beregarded as illustrative and not limiting the scope of the invention.All references cited in the present application are incorporated hereinby reference.

EXAMPLES Materials and Methods

1. Production and culture of human FGF-2-secreting cells

To produce human FGF-2-secreting fibroblast cells PS-FGF18 (FGF-18),mouse fibroblasts (NIH3T3) were stably transfected with the bicistronicplasmid pSFGF18 (6.43 kb) which was constructed on the basis of thevector pEN (43). The plasmid pSFGF18 contains the pVC-derived Sma I-NcoI fragment (44), encoding the amino-terminal secretion signal peptide(PS) of the vascular endothelial growth factor (VEGF), which wasinserted into the Sma I-Nco I restriction sites of pF18EN (45). Thevector further contains the upstream enhancer/promoter sequence of thecytomegalovirus (CMV) followed by the chimeric VEGF-hFGF-2 gene (Genbankaccession # of the human FGF-2 cDNA: M27968), the downstream internalribosomal entry site of the encephalomyocarditis virus, the neomycineresistance gene, the SV40 polyadenylation signal sequence, the IVS2bintron of the rabbit β-globin gene, and the ampicillin resistance gene.Thus, plasmid transcription gives rise to a bicistronic mRNA containingthe coding sequences of hFGF-2 and aminoglycoside phosphotransferase. Toobtain NIH3T3-P16 cells (P16), mouse fibroblasts were stably transfectedwith a modified pRPSFEN plasmid which lacks the hFGF-2 transgenesequence. Cells were selectively cultured in a humid CO₂ incubator at37° C. using 75 cm² flasks (Techno Plastic Products, Switzerland). TheDulbecco's Modified Essential Medium DMEM/F12 (growth medium; LifeTechnologies, France) was enriched by 10% decomplemented Australianfetal calf serum (FCS; Life Technologies, France) and contained apre-fabricated mixture of antibiotic-antimycotics (Life Technologies,France; final concentrations: 100 units/ml penicillin, 0.1 mg/mlstreptomycin, 0.25 mg/ml Amphotericin B) as well as 0.7 mg/ml geneticin(G418; Life Technologies, France).

2. Encapsulation and Cell Viability

Trypsinized PS-FGF18 and P16 cells were suspended in the growth mediumcontaining 10% FCS plus antibiotic-antimycotics (Life Technologies,France) and mounted by capillarity into Dialox-treated 5-cm-long AN69polymer fibers (50 kDa cut-off 300 μm external diameter, 200 μm internaldiameter; Hospal R & D, Meyzieu, France). Fibers were heat-sealed atboth ends and kept in culture for 1 day to allow cell aggregation andattachment to the polymer. Then, the capsules were segmented into 1.5mm. or 4.0 mm microcapsules and were kept in culture for at least 14days prior to transplantation. NIH3T3-P16 and NIH3T3 PS-FGF18fibroblasts were released from twenty-four 4.0-mm-long capsules bytrypsinization after 2, 15, 30, and 60 days in culture (n=6 for eachcell type and time point). Cell viability was determined by trypan blueexclusion in growth medium (0.2% Trypan blue; Life Technologies, France)using a hemocytometer. Eight 1.5 mm microcapsules filled with eitherNIH3T3-P16 or NIH3T3 PS-FGF18 cells were explanted after 45 days (n∝foreach cell type). Similarly, a microcapsule with NIH3T3-P-16 cells and amicrocapsule filled with NIH3T3 PS-FGF18 cells were explanted at 90 dayspost-transplantation. Microcapsules were trypsinized inphosphate-buffered saline (PBS) for 20 mm at 37° C., rinsed in growthmedium, and opened to release cells for trypan blue exclusion assays.

3. Biopolymer Permeability and Bioassay

Three 1.0-cm-long capsules were pre-incubated for 24 h with growthmedium containing 10% FCS plus antibiotic-antimycotics and subsequentlyfilled with 125 ng recombinant hFGF-2. Following another 24 h incubationin growth medium at 37° C., the concentration of soluble andsubstrate-bound protein released from capsules was measured by ELISA.Microcapsules with a length of 1.6 mm and 4.0 mm, which were eitherempty (n=6, 4.0 mm) or filled with NIH3T3 PS-FGF18 or NIH3T3 P16 cellsand kept in culture for 15, 30, or 60 days (n=6 per cell type and timepoint), were transferred to 6-well TPP culture plates (1microcapsule/well; Techno Plastic Products, Switzerland) containingadherent NIH3T3 fibroblasts (5×104/well). After 10 days of co-culturingin growth medium containing 10% FCS plus antibiotic-antimycotics at 37°C. 5% CO₂, the NIH3T3 cell density was determined with a hemocytometerand compared to untreated NIH3T3 fibroblast cultures. The cellproliferation rate under the experimental conditions was analyzedstatistically using the Mann-Whitney U-test.

4. hFGF-2 production determined by Western Blotting and Enzyme-LinkedImmuno-Sorbant Assay (ELISA)

Stably transfected fibroblasts were sonicated on ice (3×2 sec; Bransonsonifier 450) in 20 ml PBS plus 2% sodium dodecyl-sulfate (SDS) anddenatured in 40 ml Laemmli buffer at 85° C. for 5 min. Both recombinantbacterial hFGF-2 (10 ng) and crude cell extract proteins (5×10⁵ cells)were separated by size in a 12% denaturing SDS-polyacrylamide proteingel (Protogel; National Diagnostics, Atlanta, USA) and blotted onto anImmobilon P membrane (Millipore, France). After overnight incubation ofthe membrane with primary polyclonal rabbit anti-hFGF-2 antibodies(1/200; Santa Cruz Biotechnology, USA) in 5% fat-free milk powder/TBST(10 mM Tris-HCl pH 8, 150 mM NaCl, 0.05% (v/v) Tween-20®), the hFGF-2protein was visualized by means of an ECL kit (Amersham, France)according to the manufacturer's instruction. The secretion of hFGF-2from 106 NIH3T3 PS-FGF18 cells, cultured in 6-well TPP culture platesfor 24 h in growth medium at 37° C./CO₂, was measured by a Human FGFBasic ELISA kit (Quantikine; R&D Systems, Abingdon, UK) according to themanufacturer's instructions. Protein release from 1.0-cm-long capsuleswas determined similarly after they were filled with 125 ng recombinanthFGF-2 protein and incubated in TPP culture plates with growth mediumfor 24 h at 37° C. Detachment of hFGF-2 from the culture plate substratewas carried out at 37° C. by successive 20 min incubation steps with 2 MNaCl, 20 mM sodium acetate (pH 4.5) and 2 M NaCl, 20 mM HEPES pH 7.5.

5. Intravitreal Transplantation

1.5-mm-long microcapsules, filled with either NIH3T3-P16 or NIH3T3PS-FGF18 cells, were kept in culture for 14 days. Leak-proof capsuleswere then transplanted into the vitreous cavity of 29 globes from21-day-old RCS p+ rats (n=21 rats; kindly provided by Dr. M. M. LaVail,UCSF, San Francisco, USA). For transplantation, these rats wereanesthetized by an intraperitoneal injection of 100 mg/kg ketamine(Kétalar; Laboratoire Substantia, France). Topical anesthesia byoxybuprocaine chlorydrate (Cébésine, Laboratoire Chauvin, France) wasapplied onto the cornea to eliminate the eyelid reflex. Head and eyelidwere cleaned with 70% ethanol prior to an external canthotomy of theeye. The temporal superior region of the conjunctival tissue was cut at1 mm from the limbus. The ocular globes were perforated with a surgicalmonofilament needle (10/0). The perforation was enlarged with a surgicalscalpel to 1 mm in length to allow the intravitreal transfer of a 1.5 mmmicrocapsule filled with either NIH3T3-P16 (unable to produce and torelease hFGF-2) or NIH3T3 PS-FGF18 cells (producing and secreting therecombinant 18 kDa hFGF-2 isoform). The canthotomized eyelids wereclosed with 3 stitches using a vicryl filament (8/0). The treated eyeswere finally covered with a paste of Sterdex (Laboratoire Martinet,France) containing dexamethasone and oxytetracycline. Operated rats ofthe same experimental group were kept together for 45-90 days understandard animal housing conditions at an illuminosity between 0 I× (12hrs dark) and 150 I× (12 hrs light) with food and water being availablead libitum. Both the transplanted and the control animals wereclinically examined biomicroscopically each day.

In the first experimental series (intravitreous transplantation), fourexperimental groups of rats were constituted following a procedure ofrandomization. Group I (6 rats) and group II (4 rats): the eyes werehistologically analyzed at 90 days post-transplantation; group III (6rats) and group IV (5 rats): the eyes were studied histologically at 45days post-transplantation. In all groups, each eye was randomly chosenby the surgeon for microcapsule transplantation. In groups I and III,microcapsules containing hFGF-2-producing cells were randomly allocatedto the eyes whereas the contralateral eyes of the same rats receivedmicrocapsules containing NIH3T3 P16 cells which are unable to releaserecombinant hFGF-2. In the groups II and IV, hFGF-2-releasingmicrocapsules were randomly allocated to the eyes while thecontralateral eyes of the same rats did not receive any treatment. Oneeye of group I which received a microcapsule filled with NIH3T3 PS-FGF18cells presented endophthalmitis. In group III, analyzed at 45 dayspost-transplantation, microcapsules containing NIH3T3-PS-FGF18 cellswere randomly allocated to six eyes whereas the contralateral eyesreceived encapsulated NIH3T3 P16 cells. In group IV, one rat displayedunilateral endophthalmitis which was confined surprisingly to theuntreated contralateral eye. The treated eye of the same rat could notbe analyzed histologically due to difficulties encountered during thecryostat cutting procedure. In total, 3 eyes were excluded from thestatistical analysis. Thus, out of 42 eyes (n=21 rats) involvedinitially in our first experimental series, only 39 eyes could besubjected to detailed histological and statistical analyses. A secondexperimental series involved 5 rats: each rat received subretinaltransplantation of a microcapsule filled with either NIH3T3 PS-FGF18(n=3) or NIH3T3-P16 cells (n=2). The contralateral eyes had no surgicaltreatments. This type of implantation caused surgically induced localtissue necrosis. One of the two eyes which had a subretinaltransplantation of a microcapsule containing NIH3T3-P16 cells presentedendophthalmitis.

6. Tissue Staining and Histological Analysis

All treated rats were subjected to pupillary reflex testing andenucleated post-mortem after CO₂ asphyxia. Ocular globes were embeddedin Tissue-Tek O.C.T. compound (4583; Miles Diagnostics, Elkhart, USA),immediately frozen in dry ice-cooled isopentane, and cut on a cryotome(Leica cryocut 3000). Serial 16-mm-thick sections were mounted ontoglass slides that were pre-treated with 2% (v/v)3-aminopropyl-triethoxysilane in acetone and postfixed for 20 min with2% (w/v) paraformaldehyde in 0.1 M phosphate buffer pH 7.4 at 4° C.After toluidine blue staining and incubation for 5 min in 5% ammoniumheptamolybdate in PBS, sections were dehydrated in ethanol andcoverslipped in Eukitt (O. Kindler GmbH, Freiburg, Germany). In eacheye, the number of photoreceptor cell layers was counted on sectionsabout 50 mm apart under a light microscope at a 400-fold magnification.In each section evaluated, the number of photoreceptor cell rows in theouter nuclear layer was determined at field points being 125 mm apart.If a field point revealed a row number with more than 4 photoreceptorcell layers, it fulfilled the criterion for photoreceptor cell rescue.The field of delayed cell degeneration was determined on two-dimensionalschematic representations of the retina. Differences in the rescuedretinal area between the experimental groups which receivedmicrocapsules, filled with either NIH3T3-P16 (n=12 eyes) or NIH3T3PS-FGF18 cells (n=19 eyes), were analyzed statistically using theMann-Whitney U-test. After elimination of the four endophthalmic eyesand the one eye which was damaged during sectioning, 39 eyes out of 42(n=21 rats) were available for the counting of photoreceptor nuclei rowsin the outer nuclear layer of the rat retinas.

7. Immunohistochemistry

Ocular globes were fixed for 2 days in Davidson fixative (Prolabo,France) and embedded in parafin. Serial 5-mm-thick sections were cutwith a microtome and mounted onto DAKO ChemMATE capillary gap microscopeslides (DAKO, France). After an exposure to 56° C. for 2 days, theslides were boiled for 10 min in 800 ml 0.1 M citric acid (adjusted topH 6 with 2 N HCl) using a microwave oven (750 W). Then, the solutioncontaining the slides was left at ambient temperature during 30 minbefore the sections were rinsed in PBS. Following 5 min incubation inTBS (0.05 M Tris-HCl, 0.15 M NaCl, pH 7.6) and a rinse step in TBS plus1% bovine serum albumin (BSA), the sections were exposed to 10 ng/mlmonoclonal mouse anti-bovine basic FGF IgGs (type II; UpstateBiotechnology; Lake Placid, N.Y., USA) in DAKO ChemMATE Antibody Diluent(DAKO, France) for 1 h at room temperature. After successive washingsteps in 0.05 M Tris-HCl (pH 6) and TBS plus 1% BSA, immunostaining wascarried out with biotinylated secondary IgGs, horseradish peroxidase,and diaminobenzidine (DAB) using the ChemMATE Detection kit (DAKO,France). The DAB-stained sections were subsequently colorated withHarris' hematoxyline (Reactifs RAL, Paris, France) for 1 min and rinsedin a saturated aqueous solution of lithium carbonate (Prolabo, France).Then, the sections were dehydrated in ethanol and rinsed with tolueneand xylene before they were coverslipped in Eukitt.

Results

8. Viability of Encapsulated Fibroblasts

The viability of encapsulated NIH3T3-P16 and NIH3T3 PS-FGF18 cells wasexamined first under culture conditions. In 4.0 mm microcapsules, thesecells typically reaggregated and attached to the biomaterial AN69 within24 hours when kept in growth medium (FIG. 1(1)). As determined at 15days post-encapsulation, both cell types filled the lumen of the fibersby reaching an average density of 2.16×10⁵ cells per microcapsule (FIG.1 (2)). At this time point, an average 75% of NIH3T3 PS-FGF18 cells and67% of NIH3T3-P16 cells were viable inside the microcapsules, asdetermined by trypan blue exclusion (FIG. 2). When encapsulated cellswere cultured for 30-60 days, cell survival declined to 60-65% in NIH3T3PS-FGF18 cells and to 2% in NIH3T3-P16 cells (FIG. 2).

After 1.5 mm microcapsules with an internal volume of 0.3 ml were keptin culture for 14 days, leak-proof capsules were selected andsubsequently transplanted into the vitreous cavity of RCS rats (FIG.1(3)). Cell survival was measured by trypan blue exclusion at 45 daysand 90 days post-transplantation. 58% of NIH3T3 PS-FGF18 cells and 69%of NIH3T3-P16 cells were viable in microcapsules at 45 days. Their cellnumber varied between 2×10³ and 10⁴ cells per microcapsule. A few NIH3T3PS-FGF18 cells retained their ability to attach to the culture platesubstrate after being released from the capsules. As found in amicrocapsule explanted after 90 days in oculo, 29% of NIH3T3 PS-FGF18cells were viable. On the other hand, NIH3T3-P16 cells had changed theirmorphological appearance at this time point and had fissioned toapoptotic bodies. On toluidine blue-stained 16-mm-thick sections cutthrough microcapsules embedded in the vitreous body, a homogeneousdistribution of NIH3T3 PS-FGF18 cells was found. Whereas in transplantedmicrocapsules filled with NIH3T3-P16 cells, the cells formed aggregateswhich caused intercellular gaps.

9. Expression of hFGF-2

The expression of the hFGF-2 transgene by encapsulated and transplantedNIH3T3 PS-FGF18 cells was verified by immunohistochemistry. As seen on5-mm-thick sections through a microcapsule filled with NIH3T3 PS-FGF18cells at 90 days post-transplantation, hFGF-2-like immunoreactivity (IR)was detected in the cytoplasm of about one third of encapsulated cells(FIG. 9 (5)). Immunohistochemical staining of the inner and outernuclear layers of the retina as well as the RPE cell layer serve as apositive control for the antigen-specificity of the antibody used (FIG.9 (1)). Unlike the cytoplasmic hFGF-2-IR of encapsulated cells, theretinal cells appear to have mainly nuclear FGF-2 staining.

10. Release of hFGF-2 from microcapsules

The production of hFGF-2 in cultured NIH3T3 PS-FGF18 cells wasvisualized and quantified by Western blotting and ELISA. On immunoblots,cellular hFGF-2 has an apparent molecular weight of 18 kDa whichcorresponds in size to the recombinant hFGF-2 protein (FIG. 3). InNIH3T3-P16 cells, in turn, the concentration of the endogenous 18 kDaform of FGF-2 was below the threshold of detection. As quantified byELISA, NIH3T3 PS-FGF18 cells secrete an average of 68.7 pg±2.3 (s.e.m.;n=6) hFGF-2/106 cells within 24 h (FIG. 4). This value represents theoverall level of extracellular hFGF-2 whereas 96% of which was found toadhere to the culture plate substrate. The permeability of thebiopolymer membrane for the hFGF-2 protein was further verified in1.0-cm-long capsules. In these capsules, 2% of hFGF-2 diffused throughthe biopolymer membrane during 24 hrs. at 37° C. As determined by abioassay, the quantity of released hFGF-2 is sufficient to stimulatecell proliferation. During a co-culture period of 10 days with eitherencapsulated NIH3T3-P16 cells or empty microcapsules, NIH3T3 cells grewfrom 5×10⁴ cells to 6.5×10⁶ cells per well (FIG. 5). A similarproliferation rate was found in untreated adherent NIH3T3 cell cultures.When NIH3T3 cells were exposed for 10 days to NIH3T3 PS-FGF18-filledcapsules kept in culture for 15-60 days, a significant increase inNIH3T3 cell proliferation by 35%±2.1 (s.e.m.; 1.5 mm capsules, n=18) and58%±2.6 (s.e.m.; 4.0 mm capsules, n=18) was observed as compared toNIH3T3-P16 capsules (P=0.001, Mann-Whitney U-test).

11. Photoreceptor Cell Rescue and FGF-2-immunoreactivity

The ability of NIH3T3 PS-FGF18-filled microcapsules to delayphotoreceptor cell degeneration was studied by analyzing the number ofphotoreceptor cell layers in the RCS p+ rat retina. At postnatal day 21,the RCS retina normally shows an average number of 10 photoreceptor celllayers (FIG. 6 (1)). After 45 days, these layers are typically reducedto 1-2 rows in untreated eyes (FIG. 6 (2)). At 45 and 90 dayspost-transplantation, NIH3T3-P16 microcapsule implants caused a delayedphotoreceptor cell degeneration covering an average retinal area of 0.08mm2±0.04 and 0.025 mm2±0.03, respectively (s.e.m.; n=6; Table I).

FIG. 7 shows a graphical representation of an eye with a NIH3T3-P16microcapsule which had one of the largest surface (0.15 mm²) fulfillingthe criterion for delayed cell degeneration (described in Materials andMethods; FIG. 7 (2), Table I). In the majority of ocular bulbi withNIH3T3-P16 microcapsules as well as in all the untreated eyes, thenumber of photoreceptor cell layers was reduced to a single cell row.Eyes which received NIH3T3 PS-FGF18 microcapsules retained 5-6photoreceptor cell layers in the retina close to the implant at 45 daysas well as at 90 days post-transplantation (FIG. 6 (3)). A rescued areaof 2.08 mm²±0.72 (s.e.m.; n=10), observed at 45 dayspost-transplantation, was restricted to the retinal zone at the vicinityof the microcapsule and was significantly higher than that observed inuntreated control eyes (P=0.001, Mann-Whitney U-test; Table I). Themaximal rescue effect amounted to 4 mm² or 10% of the entire retinalsurface and represents 10 times the region covered directly by themicrocapsule (FIG. 7(1)). Similarly, an average retinal area of 0.95mm²±0.22 (s.e.m.; n=9) was rescued by 90-day-old implants. This localrescue effect is significantly lower than that found at 45 dayspost-transplantation but is significantly higher than the effect ofNIH3T3-P16 microcapsules on the survival of photoreceptor cells (P<0.05,Mann-Whitney U-test; Table I). In untreated eyes, no more than 3photoreceptor cell layers were found in the retina at both time points(FIG. 6 (4)).

Rosette formation and abnormal thickness of the retina were detected in2 out of 31 treated globes which received encapsulated NIH3T3 PS-FGF18or NIH3T3-P16 cells (FIG. 8). Furthermore, posterior cataracts wereapparent in 3 eyes with NIH3T3-P16 microcapsules as well as in 2 globeswith encapsulated NIH3T3 PS-FGF18 cells. Three treated eyes showed signsof surgically induced infection leading to ocular atrophy in theexperimental series. In 29 ocular globes, the microcapsules wereentirely intravitreal and revealed neither apparent graft rejection byintraocular macrophage infiltration nor fibrous formation around themicrocapsule. In the 2 ocular globes displaying rosettes, themicrocapsules rested upon the inner surface of the retina.

The endogenous production of FGF-2 in the RCS retina was visualized byimmunohistochemistry. In the normal retina of a 3-month-old albino RCSp-rdy+ rat, mainly nuclear FGF-2-IR was apparent in some cells of theinner nuclear layer as well as in all photoreceptor cells of the outernuclear layer (FIG. 9 (2)). Cell nuclei of RPE cells also revealedstrong immunostaining. In dystrophic 111-day-old RCS p+ rats, however,FGF-2-IR was found in some cells of the inner nuclear layer (FIG. 9(3)),but it was absent from degenerating photoreceptor cells. In severalage-matched RCS rats which received NIH3T3 PS-FGF18 microcapsules, thelocally rescued photoreceptor cells were also devoid of FGF-2-IR whereasthe inner nuclear layer still revealed some immunostained cells (FIG. 9(4)).

CLONING THE GENE ENCODING MOUSE ENDOSTATIN INTO CHO CELLS

Sequences encoding endostatin protein were amplified by PCR usingGoldstar polymerase (Eurogentec). Primers endo s:

(3′-CCGACTTTCAGGACTACTCATACTTAAGGGTACCAAA-5′) (SEQ ID NO:4) containing alinker with NcoI

and endo s his rev:

(3′GTACTGGAGAAAGAGGTTTCGGCCGTCTCCTAGCGTAGTGGTAGTGGTAGTGCACATCGGCGCCAAA-5′) (SEQ ID NO:5), containing his tag and linker withSacII were used. Mouse cDNA was used as a template.

For a 50 μl final volume, 1 μl of the cDNA template, 0.5 μg of eachprimer, 0.2 mM DNTPs, 5 μl 10× Goldstar Reaction Buffer (fromEurogentec), 1.5 mM MgCl₂ and 2.5 Units Goldstar Polymerase (fromEurogentec) was used. PCR was carried out for 30 cycles using thefollowing parameters: 94° C. denaturation for 1 minute; 62° C. annealingfor 1 minute; and 72° C. annealing for 1 minute.

The product of the amplification (540 Kb) was sequenced on both strandsusing a Perkin Elmer Kit.

After completion, the product of the amplification was gel purifiedusing Gene-Clean (Boi 101, La Jolla) and the linkers were digested withthe appropriate enzymes and then again purified using Gene-Clean.

The gene fragment was ligated into a PRCEN expression vector in fusionwith the VEGF peptide signal and tag HA (Hemaglutinine).

The resultant E.coli clones were selected and plasmid preparations(pRCEN endo) of clones were obtained and sequenced.

Stable CHO clones were obtained by transfection with pRCEN endo (2 μg)using a fuGENE™ 6 transfection reagent (Boehringer Mannheim) in a 60 mmdish containing 5 ml of MEM (Minimum essential Medium, Gibco, BRL)complemented with 5% FCS, 1% Glutamine, 1% amphotericine B and 0.5%Gentamicine). 24 hours later the cells were trypsinized and transferredinto 3 100 mm dishes in the presence of G418 (1 mg/ml). The cultureswere maintained for 2 weeks in the presence of G418, and 12 clones werepicked up and transferred into 24-well plates before cultivation inlarger dishes.

The construct of plasmid pRCEN1-Endo is shown in FIG. 10. The varioussequences used to construct this plasmid such as the peptide signalsequence VEGF, HA and endo are shown in FIG. 11.

Western immunoblotting was then performed using a conditioned medium.The cells were then precipitated with TCA and the cell lysates of thedifferent clones were analyzed by western immunoblotting usingpolyclonal anti-HA antibody (Eurogentec). The Western blot is shown inFIG. 12.

In the present invention, a novel intravitreal cell therapy approach foran experimental treatment of retinal dystrophies is disclosed. Thetherapeutic transgene hFGF-2 was transferred to the ocular bulbi viastably transfected and encapsulated NIH3T3 PS-FGF18 fibroblasts. Thesecells as well as hFGF-negative fibroblasts (NIH3T3-P16) can adhere tothe biopolymer and proliferate inside the microcapsules. Fibroblastswhich produce no or only a low level of hFGF-2 showed limited cellsurvival under encapsulation conditions. These cells typically continueto proliferate inside microcapsules under culture and in oculoconditions, grow to confluency, and eventually undergo cell apoptosis.Due to the anti-apoptotic action of FGF-2 (46), however, cell apoptosiscan be impeded by the intracellular production of FGF-2. Indeed,encapsulated NIH3T3 PS-FGF18 cells, which produce and secrete a highlevel of the 18 kDa protein isoform of hFGF-2, revealed long-term cellviability both under culture condition and inside the vitreous cavity.Due to the hydrophobic VEGF secretory signal peptide, linked to theamino-terminus of the protein, hFGF-2 secretion is favored by theendoplasmic reticulum-Golgi complex secretory pathway. As shown byimmunohistochemistry on encapsulated NIH3T3 PS-FGF18 fibroblasts at 90days post-transplantation, about 30% of these cells continue to producehFGF-2 which seems to be concentrated in the cytoplasm. Using abioassay, it was confirmed that encapsulated NIH3T3 PS-FGF18 fibroblastscan stimulate cell proliferation, with a dose-response effect, in acapsule/NIH3T3-P16 cell co-culture system. This finding demonstratedindirectly that secreted hFGF-2 can diffuse through the biopolymermembrane of the capsule.

The membrane permeability for hFGF-2 has also been observed in vivo.When transplanted into the vitreous cavity, microcapsules filled withhFGF-2-secreting fibroblasts provoked a delay of photoreceptor celldegeneration. At 45 days post-transplantation, this cell rescue effectwas dearly observed in up to 10% of the retinal surface. The averagearea rescued by 90-day-old microcapsules, however, is about half of thesize of that detected at 45 days post-transplantation. As the viabilityof encapsulated hFGF-2-producing fibroblasts gradually declines in vivo,photoreceptor cell degeneration may have resumed at 90 dayspost-transplantation.

In ocular globes with encapsulated NIH3T3-P16 cells, the photoreceptorcell rescue effect was minimal, indicating that secreted hFGF-2 isresponsible for the delay of photoreceptor cell degeneration. Thisobservation further shows that the surgical trauma caused by themicrocapsule implantation did not induce cell rescue. In contrast, inother experimental settings such as intravitreal injections of PBSversus hFGF-2, control animals displayed a significant local rescueeffect (33). Although hFGF-2-releasing microcapsules lead to a localdelay of photoreceptor cell degeneration in 66 and 111-day-old RCS p+rats, the rescued retinal photoreceptor cells had an undetectable levelof FGF-2-immunostaining. This finding is in line with a reduced level ofFGF-2 protein in overall retinal preparations of RCS rats (27). Since,after the onset of the retinal degeneration, a transient increase ofFGF-2 concentration can occur in photoreceptor cells of RCS rats ascompared to those of normal RCS rdy+ rats (47), it must be concludedthat in the experiments the rescued photoreceptor cells failed to regainthe ability of producing endogenous FGF-2. Thus, the rescue effect ofphotoreceptor cells seems to depend entirely on the presence or absenceof exogenous FGF-2 in the retina.

Under in vivo conditions the hFGF-2 protein released by the microcapsulediffuses through the vitreous humor and is presumably absorbed byganglion and Müller cells in the retina close to the microcapsule. Asrecently described, exogenous FGF-2 can stimulate the expression of theFGF-2 gene in cultured Müller cells (48). In adult RCS rats withdystrophic retinas, a significant FGF-2-IR in large cell bodies of theinner nuclear layer which may correspond to Müller cells was also found.Since these cells span the entire retina, an hFGF-2-stimulated increaseof the production and secretion of endogenous FGF-2 may help to slowphotoreceptor cell degeneration. Moreover, FGF-2 inhibits the productionof cytotoxic nitric oxide in RPE cells which can affect indirectly thesurvival of photoreceptor cells (49-51). It cannot be excluded that adirect effect of hFGF-2 on photoreceptor cells and/or RPE cells afterdiffusion throughout the different retinal layers.

As it has been shown previously, FGF-2 can potentially induce tumor-likecell proliferation (52). Although any signs of tumor formation was notfound, a few photoreceptor cell rosettes were detected in two out of the31 ocular globes analyzed. This abnormality is independent of the typeof microcapsules implanted, suggesting that the malformation is causedby the surgical trauma. Despite the local application of corticoids andantibiotics, three eyes developed endophthalmitis, two in the firstexperimental series and one in the second series. Since ocular atrophywas not restricted to eyes which received a particular type ofencapsulated cells, surgically-induced infection may be at the origin ofendophthalmitis. It was noticeable that in one eye of the firstexperimental series, endophthalmitis occurred in an untreated eye,suggesting that aseptic procedures should be improved to avoid thisiatrogenic complication. Moreover, all treated globes were devoid ofmicrocapsule-mediated fibrosis or hyperacute macrophage cellinfiltration into the vitreous cavity, indicating that the biomaterialdid not provoke any cell-mediated immune response. Within microcapsules,however, fibroblasts gradually undergo apoptosis. Since the 50 kDacut-off pore size of the AN69 biopolymer hampers the penetration ofIgMs, membrane attack complexes and C1 fraction of the complement (53),cell death inside the microcapsule was probably not provoked by the hostcomplement system. These findings indicate that the biomaterial is notimmunogenic per se and sufficiently protects hFGF-2-secretingfibroblasts against cell death mediated by immune responses.

Besides using the hFGF-2 protein, other well characterized cell linescan be used which are capable of releasing other trophic factors. Forexample, cell lines can be engineered which produce the ciliaryneurotrophic factor (CNTF) and/or the glial cell line-derivedneurotrophic factor (GDNF) which are indispensable for their ownproliferation as well as for photoreceptor cell survival.

The use of inducible promoters such as the tetracycline regulationcassette which allows to experimentally modulate transgene transcriptionis obviously an additional potential protection against unpredictableiatrogenic effects.

Furthermore, the present invention is not limited to the use of AN69.Other biomaterials which are, first of all, as efficient as AN69 interms of limiting humoral and cellular immune reactions and, inparticular, the activation of the complement system, and secondly, allowa better diffusion of therapeutic molecules can be used.

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                   #             SEQUENCE LISTING<160> NUMBER OF SEQ ID NOS: 5 <210> SEQ ID NO 1 <211> LENGTH: 206<212> TYPE: DNA <213> ORGANISM: Rattus rattus <400> SEQUENCE: 1cccgggcctc gggccgggga ggaagagtag ctcgccgagg cgccgaggag ag#cgggccgc     60cccacagccc gagccggaga gggagcgcga gccgcgccgg ccccggtcgg gc#ctccgaaa    120ccatgaactt tctgctgtct tgggtgcatt ggagccttgc cttgctgctc ta#cctccacc    180 atgccaagtg gtcccaggct gcaccc          #                   #             206 <210> SEQ ID NO 2 <211> LENGTH: 79<212> TYPE: DNA <213> ORGANISM: Rattus rattus <400> SEQUENCE: 2atgacttacc catacgatgt tccagattac gctagcttgg gtggtcatat gg#ccatggag     60 gccccgggga tccgaattc              #                  #                   # 79 <210> SEQ ID NO 3 <211> LENGTH: 558<212> TYPE: DNA <213> ORGANISM: Rattus rattus <400> SEQUENCE: 3gctcatactc atcaggactt tcagccagtg ctccacctgg tggcactgaa ca#cccccctg     60tctggaggca tgcgtggtat ccgtggagca gatttccagt gcttccagca ag#cccgagcc    120gtggggctgt cgggcacctt ccgggctttc ctgtcctcta ggctgcagga tc#tctatagc    180atcgtgcgcc gtgctgaccg ggggtctgtg cccatcgtca acctgaagga cg#aggtgcta    240tctcccagct gggactccct gttttctggc tcccagggtc aactgcaacc cg#gggcccgc    300atcttttctt ttgacggcag agatgtcctg agacacccag cctggccgca ga#agagcgta    360tggcacggct cggaccccag tgggcggagg ctgatggaga gttactgtga ga#catggcga    420actgaaacta ctggggctac aggtcaggcc tcctccctgc tgtcaggcag gc#tcctggaa    480cagaaagctg cgagctgcca caacagctac atcgtcctgt gcattgagaa ta#gcttcatg    540 acctctttct ccaaatag              #                  #                   # 558 <210> SEQ ID NO 4 <211> LENGTH: 37<212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: PCR Primer <400> SEQUENCE: 4ccgactttca ggactactca tacttaaggg taccaaa       #                  #      37 <210> SEQ ID NO 5 <211> LENGTH: 67 <212> TYPE: DNA<213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: PCR Primer <400> SEQUENCE: 5gtactggaga aagaggtttc ggccgtctcc tagcgtagtg gtagtggtag tg#cacatcgg     60 cgccaaa                  #                  #                   #          67

What we claim is:
 1. A composition comprising encapsulated cells whichproduce an endostatin or a fibroblast growth factor polypeptide, whereinsaid encapsulated cells are encapsulated with a biocompatible materialof polyacrylonitril-methysulfonate.
 2. The composition according toclaim 1, wherein said biocompatible material forms a biocompariblemicrocapsule which prevents entry of host immune cells inside saidmicrocapsules and allow release of said endostatin or said fibroblastgrowth factor outside of the microcapsule.
 3. The composition accordingto claim 2, wherein said biocompatible microcapsule is comprised ofpores having an internal diameter below 400 μm.
 4. The compositionaccording to claim 2, wherein said biocompatible microcapsule iscomprised of pores having an internal diameter below 300 μm.
 5. Thecomposition according to claim 1, wherein said encapsulated cells aremammalian cells comprising a nucleic acid encoding said endostatinpolypeptide or said fibroblast growth factor polypeptide.
 6. Thecomposition according to claim 5, wherein said mammalian cells areselected from the group consisting of fibroblasts, muscle cells,hepatocytes, neural cells, kidney cells, and MDCK cells.
 7. Thecomposition according to claim 1, wherein said composition comprises 10⁴encapsulated cells.
 8. The composition according to claim 1, whereinsaid composition comprises 10⁵ encapsulated cells.